Gas and liquid chromatography are commonly used in analytical and preparative chemistry. A typical chromatographic instrument utilizes a stationary inert porous material held in a column; a fluid containing a sample of interest is passed through the porous material. A typical liquid chromatography system includes a mobile-phase pump, a sample injector, a column, and a detector. The pump propels the mobile-phase fluid along a pathway that passes through the injector, column, and detector. The injector introduces a sample into the mobile-phase fluid prior to entry of the fluid into the column.
Distinct chemical compounds contained in the fluid often have distinct affinities for the medium held in the column. Consequently, as the fluid moves through the chromatographic column, various chemical compounds are delayed in their transit through the column by varying amounts of time in response to their interaction with the stationary porous material in the column. As a result, as the compounds are carried through the medium, the compounds separate into bands which elute from the column at different times.
Thus, the different chemical compounds in a sample solution separate out as individual concentration peaks as the fluid elutes from the column. The various separated chemicals can be detected by, for example, a refractometer, an absorbtometer, a mass spectrometer, or some other detecting device into which the fluid flows upon leaving the chromatographic column.
An ideal chromatographic signal, or chromatogram, has well-resolved peaks sitting on a baseline response that is a constant with low noise. Commonly, chromatograms are less-than ideal and contain, for example, fused peaks and a noisy baseline that has a slope and/or a curvature.
Some problems in the analysis of liquid-chromatography data relate to absorbance detection of separations during rapid solvent-gradient changes, where the change in mobile phase composition causes a curvature or slope of a chromatogram's baseline. A baseline slope or curvature can introduce difficulty in displaying very small peaks across a full chromatogram.
In general, visualization of small peaks requires expansion of the vertical (e.g., absorbance) scale. Unfortunately, baseline curvature at times renders such visualization difficult. An analyst may, for example, adjust the vertical scale so the whole of the vertical extent of the curved baseline is visible, leaving small peaks too small to see clearly. Alternatively, the analyst may, for example, expand the vertical so that one group of adjoining peaks is well-visualized, but other peaks may then reside above or below the vertical boundaries of the viewing region.
Fast chromatography systems, in particular, can experience difficulty due to baseline curvature or slope. For example, a change in mobile phase composition that causes a curvature or slope of a chromatogram's baseline may occur in fast, high resolution, very high pressure (greater than 5 kpsi, for example) reversed-phase separations; such sample separations require, for example, as little as 1 to 5 minutes to complete. During this time, the mobile phase ramps from, for example, nearly pure water to nearly pure acetonitrile. Variations in the baseline slope or curvature that are related to the change in a mobile phase composition may become more significant and apparent with the compression of a time axis that is associated with short duration separations.
Ideally, in some systems, such gradient effects are reduced, for example, through flowcell and/or optical designs. Typical strategies applied to conventional flowcells reduce gradient-induced refractive index effects by preventing rays that strike the inner walls of a flowcell from reaching a detector.
These solutions, however, generally cannot guarantee a flat baseline during a rapid gradient for both diode array and tunable single wavelength UV-visible absorbance detectors, particularly when light guiding flowcells are employed. Moreover, these solutions generally are not applicable to high peak capacity chromatography systems that utilize smaller volume flowcells while providing a long path length and high optical throughput, characteristics typically required for a high signal-to-noise measurement.
One alternative prior approach to the removal of baseline curvature or slope is suitable only for multi-wavelength detectors, such as photodiode array-based detectors. In this approach, a band of wavelengths is designated as a reference, where it is assumed that the analytes of interest do not absorb. As the separation progresses, absorbances at the analytical wavelengths are adjusted for changes in absorbance at the reference wavelength. This approach is preferably applied only when the baseline effects are the same at all wavelengths, a condition often not met using light guiding flowcells. Serious errors arise if any of the eluting compounds absorb at the reference wavelength. Moreover, noise from the reference wavelengths is added to the noise on the analytical signal.